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Introduction

Hypoxia inducible factors (HIFs) belong to the family of helix-loop-helix-PAS domain transcription factors (1,2). It has been demonstrated that there are ~150 HIF target genes (3). HIFs accelerate tumor progression and cell survival by regulating a wide variety genes that control various metabolic processes, including anaerobic metabolism (glucose transporter 1), angiogenesis (vascular endothelial growth factor), regulation of cell cycle and intracellular pH (carbonic anhydrase-9), response to DNA damage, alteration of the extracellular matrix and cell adhesion, migration, proliferation and apoptosis [p21, p27, matrix metalloproteinase (MMP)-2 and 9] (4–7). The HIF pathway in hypoxia is an important therapeutic target for reducing the size, metastatic potential and therapeutic resistance of the primary tumor (8).

There are three isoforms of the HIF-α subunit: HIF-1α, HIF-2α and HIF-3α. HIF-2 is dimerized by the HIF-1β subunit and HIF-2α subunit, and the stability and transcriptional activity of HIF-2α is accommodated by oxygen-dependent hydroxylation. In normoxic conditions, the α subunit is constitutively expressed but rapidly degraded. In a low-oxygen environment, the α subunit is stabilized and translocated to the nucleus (9,10). HIF-2α is regulated by fewer genes compared with HIF-1α; in breast adenocarcinoma MCF-7 cells, there are only a small group of hypoxia-associated genes that are associated with HIF-2α, while 80% of hypoxia-regulated genes are associated with HIF-1α, including vascular endothelial growth factor, erythropoietin and matrix metalloproteinases (11,12). Previous studies have confirmed that HIF-1 is associated with tumor progression in certain carcinomas, including breast, non-small cell lung and uterine cancer, and patients with high levels of HIF-1 have a poor response to cancer therapies (13–19). However, little is understood concerning the effect of HIF-2α in solid tumors. Previous studies have demonstrated that a cell's reaction to hypoxia is primarily regulated by HIF-1α in all cells, including breast carcinoma cells, but is regulated by HIF-2α in gastrointestinal epithelium, heart, kidney, and renal carcinoma cells (20).

RNA interference (RNAi) is a powerful mechanism for targeting post-transcriptional gene silencing, in which double-stranded RNA is successfully introduced into mammalian cells, which downregulates the expression of target genes or suppresses the replication and transcription of pathogens by degrading the homologous mRNA sequences (21). In the present study, synthesized small interfering (si) RNAs targeting the HIF-2α gene was transfected into breast adenocarcinoma MCF-7 cells using Lipofectamine® 2000 to knockdown the expression of the HIF-2α on a protein and mRNA level. Results from studies regarding the effect and impact of HIF-2α gene silencing on the cell growth and invasion potency may contribute to additional research on the HIF complex and its possible therapeutic applications. The present study investigated the correlation between HIF-2α and MMP-2 expression, and the significant role of HIF-2α in breast carcinoma cell survival and invasion.

Materials and methods

Cell lines and hypoxia treatment

The breast adenocarcinoma MCF-7 cell line was purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.), streptomycin (100 U/ml; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO2. When the cells had reached 80% confluence, they were cultured with hypoxia-mimetic agent, cobalt chloride (CoCl2; Sigma-Aldrich, St. Louis, MO, USA) at various concentrations (50, 100 and 200 µmol/l) for at 37°C in 5% CO2 for 24 h. A maximum HIF-2α accumulation was reached at 100 µmol/l CoCl2; therefore, 100 µmol/l CoCl2 was selected for use as the standard concentration for subsequent experiments (Fig. 1).

Figure 1. HIF-2α mRNA expression in human breast carcinoma MCF-7 cells. Lane 1, cells cultured under normoxic conditions (20% O2); lane 2, cells treated with 50 µmol/l CoCl2; lane 3, cells treated with 100 µmol/l CoCl2; lane 4, cells treated with 200 µmol/l CoCl2. The data are represented as the mean ± standard deviation. *P<0.05 vs. normoxic conditions. HIF-2α, hypoxia-inducible factor-2α; CoCl2, cobalt chloride; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

siRNAs and transfection

The HIF-2α siRNAs were designed according to the study by Meade et al (22) and were synthesized by Guangzhou Ruibo Biological Technology Co., Ltd. (Guangzhou, China) as follows: siRNA-1, sense 5′-GCAAAUGUACCCAAUGAUADTDT-3′ and antisense 5′-UAUCAUUGGGUACAUUUGCDTDT-3′; siRNA-2, sense 5′-CAGCAUCUUUGAUAGCAGUDTDT-3′ and antisense 5′-ACUGCUAUCAAAGAUGCUGDTDT-3′; negative control siRNA, sense 5′-CAGCAGGGUUGAUAGCAUGDTDT-3′ and antisense 5′-ACUGCCCCCAAAGAUGCUGDTDT-3æ. The MCF-7 cells were transfected with the siRNAs. Briefly, the cells were seeded into 24-well plates and cultured in RPMI-1640 medium for 24 h (at 37°C in 5% CO2) to reach 50–70% confluence. HIF-2α siRNA or the negative control siRNA were transfected into the cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were placed in a normoxic or hypoxic atmosphere for 24 h following transfection. The experimental groups were as follows: Parental cell group, MCF-7 cells without CoCl2 treatment; control group, MCF-7 cells treated with 100 µmol/l CoCl2; hypoxia + RNAi 1 group, HIF-2α siRNA1-transfected MCF-7 cells treated with 100 µmol/l CoCl2; hypoxia + RNAi 2 group, HIF-2α siRNA2-transfected MCF-7 cells treated with 100 µmol/l CoCl2.

Reverse transcription polymerase chain reaction (PCR)

Total RNA was extracted from the cells using TRIzol reagent (Gibco®; Thermo Fisher Scientific, Inc.). The cDNA was synthesized from the RNA using the Reverse Transcription System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The expressions of HIF-2α and MMP-2 were detected using the following primers: HIF-2α, forward 5′-TGAAAACAGAGTCCGAAGCC-3′ and reverse 5′-GTGGCTGACTTGAGGTTGA-3′; MMP-2, forward 5′-TTCAAGGACCGGTTCATTTGGCGGACTGTG-3′ and reverse 5′-TTCCAAACTTCACGCTCTTCAGACTTTGGTT-3′. The following glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were also used: forward, 5′-ATTCATCTCTCCTCTCCCA-3′ and reverse, 5′-GTTGGTGGTTGGTACTGT-3′. Primers were designed using the Primer Premier Software version 5 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). PCR was performed using the SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd., Dalian, China) and the ABI 9700 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and under the following conditions: 94°C for 45 sec and 55°C for 1 min for 35 cycles, with an initial denaturation step at 72°C for 10 min. The PCR products were subjected to 1.5% agarose gel electrophoresis, with GAPDH (580 bp) as an internal control. PCR products were quantified with a TotalLab Quant Phoretix 1D Pro software software (TotalLab Ltd., Newcastle Upon Tyne, UK).

Western blotting analysis

Protein was extracted from the cells using NP-40 Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein of breast carcinoma cells was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were displaced to polyvinylidene difluoride (PVDF) membranes by Pharmacia Phast gel electrophoresis system (Roche Diagnostics, Indianapolis, IN, USA). The PVDF membrane was blocked with 5% skim milk blocking buffer (Beyotime Institute of Biotechnology) for 1 h. The immunoblots were incubated with the following primary antibodies: Rabbit polyclonal anti-HIF-2α (dilution, 1:150; catalog no., ab73895; Abcam, Cambridge, UK); mouse monoclonal anti-MMP-2 (dilution, 1:500; catalog no., ab3158; Abcam); and mouse monoclonal anti-β-actin (dilution, 1:500; catalog no., ab6276; Abcam). Subsequently, the membranes were incubated at 4°C for 24 h with gentle agitation. After washing twice with phosphate-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (dilution, 1:1,000; catalog no., BA1054-1; BosterBio, Wuhan, China) and goat anti-mouse (dilution, 1:1,000; catalog no., BA1051; BosterBio) IgG secondary antibodies for 1 h at room temperature. After washing with PBS, the immuno-blotting signal was detected using a chemiluminescence system (ECL; Thermo Fisher Scientific, Inc.). The results were quantified using TotalLab version 2.0 software (Total Lab, Newcastle upon Tyne, UK). β-actin protein was used as an internal control.

Invasion assays

The invasion ability of the cells was detected using a Boyden chamber (BD Biosciences, Franklin Lakes, NJ, USA. For invasion assays, polycarbonate filters with 8-µm pore size (EMD Millipore, Billerica, MA, USA) were covered with Matrigel (Sigma-Aldrich), which was diluted to 1:20 with serum-free RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.). The polycarbonate filters were dried at room temperature for 24 h. In total, ~5×106 cells in 500 µl RPMI-1640 media were placed in the upper chamber and 400 µl RPMI-1640 medium was placed in the lower chamber. The cells were incubated at 37°C in 5% CO2 for 24 h. The cells on the bottom surface of the polycarbonate filter were removed and fixed in 4% glutaraldehyde solution and stained with hematoxylin and eosin (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Invasive cells were counted using a microscope (magnification, ×400; BX51; Olympus Corp., Tokyo, Japan). The invasion index and the cell invasion inhibition rate were calculated as previously described (23). Briefly, the invasion index was defined as the number of cells that migrated through the 8-µm pores of the filter in the experimental group divided by the number of cells that migrated through the filter in the control group × 100. The cell invasion inhibition rate was equal to the number of cells that migrated through the filter in the control group minus the number of cells that migrated through the filter in the experimental group divided by the number of cells that migrated through the filter in the control group × 100.

Statistical analysis

The data are presented as the mean ± standard deviation. Experiments were performed in triplicate. All statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Independent sample t-test was used to determine the statistical significance between the means. P<0.05 was considered to indicate a statistically significant difference.

Results

Hypoxia mimetic CoCl2 specifically elevated HIF-2α mRNA levels

To investigate whether the expression of HIF-2α is regulated by a hypoxic environment, the mRNA levels of HIF-2α were compared in cells that were cultured under normoxic (20% O2) and hypoxia-mimetic (CoCl2) conditions. After a 24 h incubation, HIF-2α mRNA levels clearly increased in the cells that were treated with CoCl2 (100 µmol/l). It has been established that CoCl2 treatment can prevent oxygen signaling in cells, leading to chemical hypoxia. The results revealed that the hypoxic effect increased in severity as CoCl2 dose increased from 50 to 100 µmol/l. This indicates that CoCl2 induces hypoxia in a dose-dependent manner. In addition, the expression of HIF-2α also increased in a dose-dependent manner. Notably, the toxicity of CoCl2 increased in a dose-dependent manner and thus at high doses this led to severe hypoxia and subsequent cell death. As a result, the expression of HIF-2α was decreased. By contrast, normoxic conditions (20% O2) had only a slight effect on HIF-2α transcription (Fig. 1). De novo transcription of GAPDH was similar among treatment groups. These results suggest that exposure of CoCl2 (100 µmol/l) promoted HIF-2α transcription, evidenced by the increase in HIF-2α mRNA levels. A CoCl2 concentration of 100 µmol/l was used in all subsequent experiments.

Effects of HIF-2α knockdown on the expression of MMP-2 in MCF-7 cells

The association between HIF-2α expression and its target gene MMP-2 was analyzed in MCF-7 cells using siRNAs against HIF-2α. As expected, HIF-2α siRNA markedly reduced the mRNA and protein levels of the targeted HIF-2α subunit (Fig. 2). Subsequently, the effects of HIF-2α knockdown on CoCl2-mediated induction of MMP-2 expression was analyzed. As shown in Fig. 3, MMP-2 protein and mRNA levels were downregulated significantly in MCF-7 cells transfected with HIF-2α siRNA compared with the control group (P=0.033). Overall, these data demonstrated that hypoxia induces HIF-2α expression at mRNA and protein levels in MCF-7 cells, and HIF-2α siRNA inhibited MMP-2 expression in MCF-7 cells.

Figure 2. Effects of HIF-2α knockdown on the expression of HIF-2α protein and mRNA in breast adenocarcinoma MCF-7 cells. Lane 1, cells cultured under normoxic conditions (20% O2); lane 2, cells treated with 50 µmol/l CoCl2; lane 3, cells treated with 100 µmol/l CoCl2; lane 4, cells treated with 200 µmol/l CoCl2. The data are represented as the mean ± standard deviation. *P<0.05 vs. parental cell group. HIF-2α, hypoxia-inducible factor-2α; siRNA, small interfering RNA; RNAi, RNA interference; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. Effects of HIF-2α knockdown on the expression of target gene MMP-2 protein and mRNA in breast adenocarcinoma MCF-7 cells. Lane 1, cells cultured under normoxic conditions (20% O2); lane 2, cells treated with 50 µmol/l CoCl2; lane 3, cells treated with 100 µmol/l CoCl2; lane 4, cells treated with 200 µmol/l CoCl2. The data are represented as the mean ± standard deviation. *P<0.05 vs. parental cell group. HIF-2α, hypoxia-inducible factor-2α; MMP, matrix metalloproteinase; siRNA, small interfering RNA; RNAi, RNA interference; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Effects of HIF-2α siRNAs on cancer cell invasion

The effects of HIF-2α siRNAs on cell migration were assessed using a Boyden chamber assay, which has been modified. As shown in Fig. 4, HIF-2α siRNA significantly inhibited cell migration compared with the control group (hypoxia + RNAi 1, P<0.006; hypoxia + RNAi 2, P=0.004). By contrast, the migratory ability of the control group cells were significantly increased compared with the hypoxia + RNAi groups (hypoxia + RNAi 1, P<0.006; hypoxia + RNAi 2, P=0.004) (Fig. 4). There was no difference on the migratory capabilities of the hypoxia + RNAi groups compared with the parental cell group (P=0.176). These data suggest that HIF-2α was a positive regulator for cell invasion and HIF-2α siRNA may inhibit the invasive capacity of MCF-7 cells (Table I).

Figure 4. Suppression effects of hypoxia-inducible factor-2α small interfering RNA on the invasion potency of breast adenocarcinoma MCF-7 cells. Migrated cells were stained with hematoxylin and eosin from the (A) parental cell, (B) control, (C) hypoxia + RNAi 1 and (D) hypoxia + RNAi 2 groups. A significant reduction in the number of invaded cells indicated a decrease in the invasive potency of the treated cells. RNAi, RNA interference.

Table I. Effects of HIF-2α small interfering RNA on the invasion potency of human breast adenocarcinoma MCF-7 cells.

Table I.

Effects of HIF-2α small interfering RNA on the invasion potency of human breast adenocarcinoma MCF-7 cells.

Group	No. of cells	Invasion index, %	Cell invasion inhibition rate, %
Parental cell	16.63±11.84a	59.06a	40.94a
Control	28.16±10.16	100.00	−
Hypoxia + RNAi 1	14.44±11.12a	51.28a	48.72a
Hypoxia + RNAi 2	13.86±15.12a	49.22a	50.78a

{ label (or @symbol) needed for fn[@id='tfn1-ol-0-0-4471'] } The data are presented as the mean ± standard deviation.

a P<0.05 vs. control group. HIF-2α, hypoxia inducible factor-2α; siRNA, small interfering RNA; RNAi, RNA interference.

Discussion

Hypoxia is common in numerous types of solid tumors, where tumor cells proliferate rapidly and form large solid tumor masses, leading to obstruction and compression of the blood vessels surrounding these masses. These abnormal blood vessels often do not function properly, resulting in a poor O2 supply to the center tumor regions. Tumor cells in this hypoxic region begin to adapt these low oxygen tension conditions by activating several survival pathways, including the HIF pathway (22,24,25). The responses of cells to hypoxia are mediated primarily through HIFs, which are critical mediators in the cellular and systemic hypoxia response to low oxygen levels via the accommodation of numerous genes that are induced by hypoxia (14,26–28). The activation of the HIF transcription factor is the most commonly identified pathway activated by hypoxic cells in harsh microenvironments. Activated HIFs exhibit a crucial role in adaptive responses of tumor cells to changes in O2 levels via transcriptional activation of >100 downstream genes which regulate vital biological processes required for tumor survival and progression. Examples include genes involved in glucose metabolism, cell proliferation, migration and angiogenesis (29). Currently, the role of HIF-2α in certain solid tumors is unclear.

The present study investigated the association and significance between the expression of HIF-2α and clinical features of breast carcinoma tissue. The author's previous histological studies have demonstrated that a higher expression of the HIF-2α protein is associated with breast cancer invasion and metastasis (30). The aim of the present study was to reveal whether HIF-2α has effects on the invasion potency of human breast carcinoma MCF-7 cells, and to reveal a possible mechanism for this function under hypoxia by using the RNAi method.

CoCl2 prevents HIF-1α from binding to prolyl hydroxylases, so that they are not hydroxylated and are subsequently degraded by proteasomes (31). CoCl2 has been used in in vivo and in vitro studies to generate hypoxia by inhibiting HIF-1 specific prolyl-hydroxylase and occupying its iron-binding site, leading to impaired binding of the von Hippel-Lindau protein with HIF-1α, subsequently preventing HIF-1α proteasomal degradation (32). Since CoCl2 stabilizes the α subunits of HIFs, the transcription of HIF-targeted genes may be induced by HIFs in spite of the presence of oxygen (4,33). CoCl2 has been successfully used to mimic hypoxia in in vivo and in vitro experimental studies (34). In the present study, a hypoxia model was successfully established through CoCl2 treatment of MCF-7 cells. The present results demonstrated that CoCl2 treatment induces HIF-2α transcription and translation and increases the mRNA and protein expressions of HIF-2α. Additionally, transfection with HIF-2α siRNA inhibited the expression of HIF-2α mRNA and protein, which indicated that the silencing effect of siRNA on HIF-2α was successful. Furthermore, the present experiments revealed that HIF-2α siRNA inhibited breast cancer cell invasion abilities; basement membrane assays reveled that there were a decreased number of invading HIF-2α siRNA transfected MCF-7 cells under hypoxic conditions in vitro compared with control cells. This finding suggests that HIF-2α may be important in MCF-7 cell survival and invasion. These results are similar to a previous study that revealed that HIF-2α promotes tumor progression in Von-Hippel-Lindau-defective renal carcinoma cells (12). By contrast in other tumor types, HIF-2α has an important role as a tumor suppressor; embryonic stem cells are deficient in HIF-2α and display enhanced growth in ovarian tumors (35). In addition, a high-expression of HIF-2α may suppress tumor growth in brain glioma cells (36).

In the present study, in order to verify whether HIF-2α affects MMP-2 expression, siRNA against HIF-2α was used to downregulate the expression of HIF-2α in MCF-7 cells. The results indicated that the expression of MMP-2 mRNA and protein decreased once MCF-7 cells were transfected with HIF-2α siRNA under hypoxic conditions. Collectively, the present findings demonstrate that the regulation of MMP-2 by hypoxia leads to increased invasion of breast cancer cells, which facilitates metastasis.

In conclusion, the present study has demonstrated for the first time, to the best of our knowledge, that HIF-2α is endogenously expressed in eukaryotic cells. In a hypoxia-tolerant tumor cell line (MCF-7) HIF-2α is involved in tumor cell invasion in vitro, which confirms the present author's hypothesis: HIF-2α facilitates cancer cell metastasis in hypoxic environments. Collectively, the present results provide a novel mechanism that HIF-2α signaling is important for cancer development and cancer cell survival by altering the expression of the downstream targets, including MMP-2. Notably, the present study revealed that siRNA targeting of HIF-2α may be a viable approach in the treatment of malignant diseases.

Acknowledgements

The present study was supported by the University Key Teacher Grant from the Ministry of Education of Henan (Zhengzhou, China; grant no. 2012GGJS-136) and Scientific Research Fund of Xinxiang Medical University (Xinxiang, China; grant no. 2014QN113).

References